Project Summary/Abstract The innate immune response is crucial for controlling infection by human pathogens. However, over-activation of the innate immune response can cause chronic inflammation that leads to human diseases, such as cancers and autoimmune disorders. To better understand and treat these diseases, developing a deeper understanding of how the innate immune system functions is paramount. In particular, the mechanisms that lead to global host shut-off of translation in response to double-stranded RNA (dsRNA), while allowing the expression of dsRNA- induced antiviral and pro-inflammatory mRNAs has remained an incompletely understood aspect of the innate immune response. Assessment of the potent antiviral endoribonuclease, ribonuclease L (RNase L), at the single-cell level revealed that it is the primary driver of translational arrest and functions by promoting rapid and widespread turnover of mRNAs. This is a significant shift in the understanding of dsRNA-induced translational arrest, as it would permit translation of mRNAs that are not degraded by RNase L. Consistent with this, the mRNA of the potent antiviral interferon-b (IFN-b) cytokine escapes RNase L-mediated mRNA turnover, potentially allowing for translation of the IFN-b mRNA. Based on these preliminary findings, this application proposes to test the hypothesis that widespread RNase L- mediated mRNA turnover functions to preferentially promote translation of antiviral mRNAs that are resistant to RNase L-mediated mRNA turnover. These findings may provide novel insights into RNase L-mediated translational arrest and antiviral gene expression that will have translational importance for understanding and treating human disease associated with dysregulation of the innate immune response. Aim 1: High-throughput sequencing and single-molecule fluorescent in situ hybridization (smFISH) will be used to identify mRNAs in addition to the IFN-b mRNA that are resistant to RNase L-mediated mRNA turnover. Aim 2: Targeted mutagenesis, chimeric mRNAs, and heterologous promoters, will be used to determine the mechanistic basis by which RNase L resistant mRNA escape RNase L-mediated mRNA turnover. Aim 3: Single-cell analysis of mRNA expression and protein translation in conjunction with ribosomal profiling will be performed to determine if RNase L-mediated mRNA promotes the translation of RNase L resistant mRNAs. Completion of these aims will determine the breadth of mRNAs resistant to RNase L-driven mRNA turnover, determine the mechanism(s) by which mRNAs escape RNase L-mediated mRNA turnover, and provide a novel mechanism by which RNase L regulates antiviral gene expression during the innate immune response. !